This proposal explores the hypothesis that serine proteases have a key role in lymphocyte-mediated lysis. Three lines of evidence support this concept: 1) inclusion of protease inhibitors in NK and T cell assays blocks killing; 2) lymphocytes have proteases in their cytotoxic granules; and 3) genes for three different proteases are preferentially expressed by cytotoxic lymphocytes. Even though protease and cytolytic proteins are both found in high density cytoplasmic granules of lymphocytes, a function for the granule proteases has not been demonstrated by other investigators. Several discoveries from the Hudig laboratory reconcile the other data and indicate that the proteases do function in cytotoxicity. Very reactive protease inhibitors block granule-mediated lysis, whereas less reactive inhibitors do not. With more sensitive substrates, they have found that there are chymotrysin-like proteases (chymases) as well as trysin-like proteases (tryptases) in the cytotoxic granules. When the proteases are inhibited, granule-mediated lysis is either delayed or completely inhibited. Their data also indicate that the protoeases may have important functions in the processing of granule proteins and that these functions occur prematurely during standard preparation of cytotoxic granules Inclusion of selected protease inhibitors in the preparation of granules can arrest cleavage of large proteins and block subsequent cytotoxicity. In this grant, they will purify and characterize the chymases and tryptases. Next they will determine which proteases are important to lysis using purified proteases to restore cytolytic activity to inhibited granules. Then they will identify the natural substrates of these proteases. These studies should indicate the first functional role for any chymase and elucidate a new serine protease cascade that initiates lymphocyte-mediated lysis. Control of this cascade could prevent acute graft rejection and other immunopathological phenomena that are mediated by cytotoxic lymphocytes.